As indicated above, the MN gene and protein are known by a number of alternative names, which names are used herein interchangeably. The MN protein was found to bind zinc and have carbonic anhydrase (CA) activity and is now considered to be the ninth carbonic anhydrase isoenzyme—MN/CA IX or CA IX [22]. According to the carbonic anhydrase nomenclature, human CA isoenzymes are written in capital roman letters and numbers, whereas their genes are written in italic letters and arabic numbers. Alternatively, “MN” is used herein to refer either to carbonic anhydrase isoenzyme IX (CA IX) proteins/polypeptides, or carbonic anhydrase isoenzyme 9 (CA9) gene, nucleic acids, cDNA, mRNA etc. as indicated by the context.
The MN protein has also been identified with the G250 antigen. Uemura et al. [23] states: “Sequence analysis and database searching revealed that G250 antigen is identical to MN, a human tumor-associated antigen identified in cervical carcinoma (Pastorek et al., 1994).”
Zavada et al., International Publication No. WO 93/18152 (published Sep. 16, 1993) and U.S. Pat. No. 5,387,676 (issued Feb. 7, 1995) describe the discovery of the MN gene and protein. The MN gene was found to be present in the chromosomal DNA of all vertebrates tested, and its expression to be strongly correlated with tumorigenicity. In general, oncogenesis may be signified by the abnormal expression of CA IX protein. For example, oncogenesis may be signified: (1) when CA IX protein is present in a tissue which normally does not express CA IX protein to any significant degree; (2) when CA IX protein is absent from a tissue that normally expresses it; (3) when CA9 gene expression is at a significantly increased level, or at a significantly reduced level from that normally expressed in a tissue; or (4) when CA IX protein is expressed in an abnormal location within a cell. WO 93/18152 further discloses, among other MN-related inventions, MN/CA IX-specific monoclonal antibodies (MAbs), including the M75 MAb and the VU-M75 hybridoma that secretes the M75 MAb. The M75 MAb specifically binds to immunodominant epitopes on the proteoglycan (PG) domain of the MN/CA IX proteins.
Zavada et al., International Publication No. WO 95/34650 (published Dec. 21, 1995) provides in FIG. 1 the nucleotide sequences for a full-length MN cDNA [SEQ ID NO: 1] clone isolated as described therein, and the amino acid sequence [SEQ ID NO: 2] encoded by that MN cDNA. WO 95/34650 also provides in FIG. 6 the nucleotide sequence for the MN promoter. Those MN cDNA, promoter and amino acid sequences are incorporated by reference herein.
Zavada et al., International Publication No. WO 03/100029 (published Dec. 4, 2003) discloses among other MN-related inventions, MN/CA IX-specific MAbs that are directed to non-immunodominant epitopes, including those on the carbonic anhydrase (CA) domain of the MN/CA IX protein. An example of such a MN/CA IX-specific MAb is the V/10 MAb, secreted from the V/10-VU hybridoma
The MN protein is now considered to be the first tumor-associated carbonic anhydrase isoenzyme that has been described. The carbonic anhydrase family (CA) includes eleven catalytically active zinc metalloenzymes involved in the reversible hydration-dehydration of carbon dioxide: CO2+H2OHCO3−+H+. CAs are widely distributed in different living organisms. The CAs participate in a variety of physiological and biological processes and show remarkable diversity in tissue distribution, subcellular localization, and biological functions [24, 25, 26]. Carbonic anhydrase IX, CA IX, is one of the most recently identified isoenzymes [22, 27]. Because of the CA IX overexpression in transformed cell lines and in several human malignancies, it has been recognized as a tumor-associated antigen and linked to the development of human cancers [8, 16, 28].
CA IX is a glycosylated transmembrane CA isoform with a unique N-terminal proteoglycan-like extension [22]. Through transfection studies it has been demonstrated that CA IX can induce the transformation of 3T3 cells [22]. Recent studies have revealed that CA IX not only participates in cell adhesion, but also can be induced in hypoxia via the HIF-1 protein binding to the hypoxia-responsive element of the MN promoter [29, 30]. The transcription of the MN gene is negatively regulated by wild-type von Hippel-Lindau tumor suppressor gene in renal cell carcinoma cells [31]. The protein product of the von Hippel-Lindau tumor suppressor gene interacts with the ubiquitin ligase complex that is responsible for targeting HIF-1α for oxygen-dependent proteolysis [32, 33]. Thus, low levels of oxygen lead to stabilization of HIF-1α, which in turn leads to the increased expression of MN [30]. Areas of high expression of MN in cancers are linked to tumor hypoxia as reported in many cancers, and incubation of tumor cells under hypoxic conditions leads to the induction of MN expression [30, 34-38].
Many studies, using the MN-specific monoclonal antibody (MAb) M75, have confirmed the diagnostic/prognostic utility of MN in diagnosing/prognosing precancerous and cancerous cervical lesions [16, 39, 40]. Immunohistochemical studies with the M75 MAb of cervical carcinomas and a PCR-based (RT-PCR) survey of renal cell carcinomas have identified MN expression as closely associated with those cancers and confirm MN's utility as a tumor biomarker [16, 39, 41]. In various cancers (notably uterine cervical, ovarian, endometrial, renal, bladder, breast, colorectal, lung, esophageal, head and neck and prostate cancers, among others), CA IX expression is increased and has been correlated with the microvessel density and the levels of hypoxia in some tumors [34, 35].
In tissues that normally do not express MN protein, CA IX positivity is considered to be diagnostic for preneoplastic/neoplastic diseases, such as, lung, breast and cervical precancers/cancers [36-38], among other precancers/cancers. Very few normal tissues have been found to express MN protein to any significant degree; those MN-expressing normal tissues include the human gastric mucosa and gallbladder epithelium, and some other normal tissues of the alimentary tract [42-44].
Renal cell carcinoma (RCC), which accounts for 3% of all adult malignancies, is the most lethal of the urologic cancers [1]. RCC is, in the US, the ninth leading cause of cancer mortality, with 35,000 new cases and more than 12,000 deaths predicted in 2004 [2]. The incidence of RCC has increased since the 1970s, largely owing to a more prevalent use of ultrasonography and computerized tomography for the evaluation of a variety of abdominal and gastrointestinal complaints [3]. For RCC, the best available prognostic indicator is stage, but the current prognostic factors: Fuhrman grade, and performance status, as well as stage, are insufficient to predict patient outcome and cancer aggressiveness [4-6]. Identification of biomarkers that provide further prognostic information would thus be vital for defining optimal treatment and outcomes.
As indicated above, previous studies have shown that MN, a member of the carbonic anhydrase family, is induced constitutively in certain tumor types but is absent in most normal tissues [7-10]. Furthermore, previous immunobiochemical studies of malignant and benign renal tissues revealed that MN is also highly expressed in RCC, suggesting that MN expression is a useful diagnostic biomarker [11, 12, 15-17]. Bui et al. [17] state that another biomarker Ki67 when used with CA IX in RCC highly predicts survival. In addition, Zavada et al. [18] discovered a soluble form of CA IX in the body fluids (urine and CA IX serum) of RCC patients. [See also, Zavada et al., International Publication No. WO 03/100029 (published Dec. 4, 2003).]
Disclosed herein are methods wherein MN expression is shown to be useful as a prognostic marker for RCC, and particularly renal clear cell carcinoma (CCC). CCCs comprise up to about 85% of RCCs. The experiments disclosed herein support the promising significance of MN as a molecular marker in RCCs, and particularly CCCs. Low MN expression in RCCs, particularly CCCs, was found to be a poor prognostic factor, and conversely high MN expression was found to be a good prognostic factor. MN expression is disclosed herein to be the best prognostic factor when compared with T stage and Fuhrman grade. Decreased MN expression is disclosed herein to be independently associated with poor survival.
The prognostic methods of this invention use 50% MN/CA IX/CA9 expression as the cut-off between better and poorer prognoses for RCCs/CCCs. Liao et al. [12] (at page 2828) described MN/CA IX immunostaining patterns “as diffuse when >50% of the cells stained and focal when ≦50% of the cells stained.” Whereas the instant inventive methods use 50% as the cut-off value, Bui et al. [15] (at page 4) found by survival tree analysis of MN/CA IX immunostaining scoring information from tissue arrays “that a staining percentage of 85% was an ideal cutoff for stratification for patient survival.” [See also, Bui et al. International Publication No. WO 03/089659 (published Oct. 30, 2003).]
The prognostic methods of this invention can be used to predict clinical outcome and tumor behavior. The prognostic methods disclosed herein detect and/or quantitate levels, extent and/or intensity of MN expression, and can identify high-risk RCC/CCC patients who could benefit from adjuvant immunotherapy and MN/CA IX/CA9-targeted therapies, among other appropriate therapies.
Preliminary data from the Bui et al. [15] study indicate a relationship between MN/CA IX and immunotherapy response. Similarly, therapies based on monoclonal antibodies to MN/CA IX or immunotherapy with MN/CA IX-based RCC vaccine [15, 19-21], as well as vectors that encode a cytotoxic protein/polypeptide and/or cytokine operatively linked to the MN gene promoter or a MN promoter fragment having promoter activity [as disclosed, for example, in Zavada et al., International Publication No. WO 00/24913 (published May 4, 2000)], preferably a MN promoter fragment comprising a hypoxia responsive element (HRE), preferably the MN HRE, can also be considered according to the level and/or extent of MN/CA IX/CA9 expression in a RCC/CCC patient sample.